Method and Apparatus for Pyrolysis-induced Cleavage in Peptides and Proteins

ABSTRACT

A method and apparatus for conducting the rapid pyrolysis of peptides, proteins, polymers, and biological materials. The method can be carried out at atmospheric pressures and takes only about 5 to 30 seconds. The samples are cleaved at the C-terminus of aspartic acid. The apparatus employs a probe on which the sample is heated and digested components analyzed.

The United States Government has rights in this invention under NationalInstitutes of Health-National Center for Research Resource(R15-RR020354-01A1) and United States Department of Agriculture (USDAGrant #448800).

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Applicant hereby incorporates by reference material submitted viaWeb-EFS in the following file:

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BACKGROUND OF THE INVENTION

This application is a divisional application of U.S. patent applicationSer. No. 12/371,893, filed Feb. 16, 2009, which is acontinuation-in-part of U.S. patent application Ser. No. 12/307,538,filed Jan. 5, 2009, which is the National Phase Entry of PCT ApplicationNo. PCT/US2007/015444, filed Jul. 5, 2007, which claims priority to U.S.Provisional Application Ser. No. 60/818,858 filed on Jul. 6, 2006

The invention relates generally to pyrolysis-induced cleavage ofpeptides and proteins and, more specifically, to a simple andsite-specific nonenzymatic method based on pyrolysis has been developedto cleave peptides and proteins.

Pyrolytic cleavage is found to be specific and rapid as it induced acleavage at the C-terminal side of aspartic acid, at the N-terminal sideof csyeine and at disulfide bonds in the temperature range of 220-250°C. in 10 s. Electrospray ionization (ESI) mass spectrometry (MS) andtandem-MS (MS/MS) were used to characterize and identify pyrolysiscleavage products, confirming that sequence information is conservedafter the pyrolysis process in both peptides and protein tested. Thissuggests that pyrolysis-induced cleavage at aspartyl residues can beused as a rapid protein digestion procedure for the generation ofsequence-specific protein biomarkers.

Protein digestion along with either peptide mass mapping orsequence-specific mass spectra forms part of a powerful bottom-up methodfor protein identification and characterization. This approach has beenmade possible by advances in both mass analyzer designs and the adventof new ionization techniques like matrix-assisted laserdesorption/ionization (MALDI) and electrospray ionization (ESI).Digestion of proteins into peptides is usually carried out by enzymaticaction, commonly tryptic, along with chemical methods like CNBr cleavageat methionine and oxidative chemical cleavage at tyrosine and trytophan.Even though these methods provide the required site-specificity forsuccessful database search and protein identification, they depend onrelatively slow enzymatic activity or require time-consuming or laborintensive procedures. Moreover, tryptic-based approaches may not beparticularly suited for proteins lacking arginine and/or lysine aminoacids or non-soluble proteins. In addition, for applications requiringautomated and field-portable instrumentation and using proteomic-basedanalyses, approaches using enzymatic digestion may add to the complexityand cost of the final field-portable device. It is with this focus onautomation and miniaturization of the sample preparation step forbottom-up proteomic analyses for microorganism detection (i.e.,biodetection) that our laboratory is developing rapid reagentlessapproaches for site-specific cleavage of peptides and proteins based onpyrolysis, electrochemical oxidation, and microwave-heated mild acidhydrolysis.

Pyrolysis has been widely used as a sample preparation step in theanalysis of low molecular weight volatile products by mass spectrometry.More recently, however, the focus has been shifted to the analysis ofnonvolatile pyrolysis products of biological and synthetic polymers byMALDI-MS.

Besides offering the ability to analyze the intact synthetic polymermolecules, ESI and MALDI allow the analysis of the non-volatilepyrolysis products of these compounds. MALDI-MS is particularly wellsuited for the analysis of high molecular weight mixtures and complexsynthetic polymer compounds due to the predominant singly charged natureof the signals generated. The use of MALDI-MS to study non-volatilepyrolysis products was first demonstrated with the analysis of pyrolyticproducts of segmented polyurethane. This study identified several seriesof oligomeric non-volatile products over the mass range ˜800-10,000 Da,including linear and cyclic polyester oligomers. MALDI-MS was alsoemployed to study low-temperature pyrolysis products from poly(ethyleneglycol). This last study found that the dominant oligomeric products hadhydroxyl and ethyl ether end groups, while at higher temperatures,methyl ether and vinyl ether end groups became more abundant in thepyrolyzates. Other studies have also used MALDI-MS for the study ofthermal oxidative degradation of nylon-6 and the thermal degradation ofaromatic poly(carbonate) polymers in the temperature range of 300-700°C. Pyrolysis was also combined with MALDIMS to study the non-volatilepyrolysis products of poly-amino acids and a small protein pyrolyzed ina nitrogen atmosphere and at temperatures ranging from 245 to 285° C. Inthis last study, the pyrolysis products were extracted and analyzed byMALDI-MS and it was hypothesized that the amino acid chains undergodehydration through the formation of cyclic oligopeptides. In addition,the use of ESI-MS for the analysis of nonvolatile pyrolysis products wasdemonstrated with the pyrolysis of dimethylamphetamine and the analysisof thermal decomposition of three common pharmaceuticals: acetaminophen,indomethacin, and mefenamic acid. In all these studies, however, samplepreparation was required and involved dissolving and extracting thenon-volatile residues with appropriate solvents (ESI) or mixing withmatrices (MALDI). This sample pre-processing step increases analysistime and could possibly affect the analysis by introducing a samplingbias and consequently not detecting important products. The introductionof ambient MS techniques has brought a new dimension in massspectrometric measurements as they allow the analysis of samples intheir native environment. To date, a number of ambient ionizationmethods for MS analysis have been introduced, but most notably aredirect analysis in real-time (DART) and desorption electrosprayionization (DESI). Of interest to this investigation is the ability ofDESI to ionize compounds from surfaces with a mechanism similar toconventional ESI and its applicability to analytes of a wide range ofmolecular weights. These analytes include, but are not limited to,pharmaceuticals and controlled substances, peptides and proteinsexplosives, clinical samples, intact tissues, synthetic polymers andbacteria. DESI is a rapid desorption/ionization source for MS andrequires little to no sample preparation. DESI is carried out bydirecting aerosolized and electrosprayed charged droplets and ions ofsolvent onto the surface to be analyzed. The charged droplets impact onthe surface and “pick up” available soluble molecules. These chargeddroplets subsequently “bounce” at a lower angle towards the MS inlet andyield gaseous ions of the compound in an analogous mechanism to that inESI. Hence, DESI yields mass spectra similar to those obtained by ESIwhich are characterized by multiply charged ions and are amenable fortandem mass analysis (MS/MS). However, it is reasonable to assume thatthe nature and polarity of the DESI solvent can be varied to affectsampling of pyrolysis products during the surface pick up step of theDESI process.

SUMMARY OF THE INVENTION

The present invention consists of heating a protein sample defined as apure protein, a mixture of proteins, whole microorganisms or intacttissue, to pyrolytic temperatures in a short period of time. Preferably,the sample is heated to between about 180° C. and about 250° C., andmost preferably to between about 210° C. and 230° C., in a period ofbetween about 5 seconds and about 30 seconds, and most preferably inabout 10 seconds. This can be carried out under atmospheric conditions.The pyrolosis can be carried out by any method that provides sufficientheat transfer to the sample, including but not limited to laserradiation, microwave radiation, membrane heaters, ovens, and stoves.

The present invention in preferred embodiments consists of the use ofpyrolysis as a sample preparation technique by applying pyrolysis as asite-specific peptide and protein cleavage method. This methodology isfound to specifically induce hydrolysis at the C-terminus of theaspartic acid residue, at disulfides bonds, and at the N-terminaus ofcysteine in a polypeptide chain in less than 10 seconds. Peptidescontaining aspartic acid were tested along with the protein lysozyme.Tandem MS (MS/MS) results confirm cleavage at the C-terminus of asparticacid.

An alternative embodiment of the present invention consists of anon-probe pyrolyzer interfaced to a desorption electrospray ionization(DESI) source as an in situ and rapid pyrolysis technique to investigatenon-volatile pyrolytic residues by MS and MS/MS analyses. The techniqueis useful in sample analysis, including the analysis of biologicalsamples and synthetic polymers.

The purpose of this invention is the rapid and non-enzymatic of peptidesand proteins at specific amino acid positions with rapid heating. Theinvention can be used in proteomic applications to where the purpose isto identify the original protein. The invention being described hereachieves the level of site-specificity, is very rapid and uses noenzymes.

The invention has advantages over the enzymatic approach in that it israpid and inexpensive. The invention performs the digestion in 10seconds as compared to the several hours to overnight incubationrequired for the enzyme approach. Moreover, the approach can be easilyautomated via an electronic circuit. This approach is also veryinexpensive as it requires simple hardware and consumes no reagents.

The invention has direct applications to proteomics research, spanningfrom the health care industry, medical research, homeland security(bioweapons detection). It can be applied to techniques to identifyproteins, mixtures of proteins, or the source of proteins as in theidentification of microorganisms.

The advantages of this methodology are its fast speed, simplicity andlow cost of the device, amino acid site-specificity, low chemical noise,and easy interfacing to MS instrumentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E show the full scan mass spectra of peptide A before pyrolysis(A) and after pyrolysis at different temperatures (B-E).

FIG. 2 is the tandem mass spectrum of Angiotensin II pyrolysis productat m/z 931.6.

FIGS. 3A and 3B are graphs of the ESI-mass spectrum of pyrolysisproducts of the VIP (1-12) peptide showing site-specific cleavage at thetwo aspartic acid sites (3A) and the ESI-mass spectrum of pyrolysisproducts of the VSV-G peptide (3B).

FIGS. 4A and 4B are graphs of the tandem mass spectra of pyrolysisproducts of the VIP (1-12) peptide, confirming their sequences.

FIGS. 5A and 5B are graphs of the MALDI-mass spectrum of pyrolysisproducts of the protein lysozyme (14 kDa), indicating the peptideproduct detected (5A) and the ESI-tandem mass spectrum of the precursorion at m/z 1201.6 (5B), confirming that sequence information ispreserved after protein pyrolysis.

FIG. 6(A) is a diagrammatical view of the on-probe pyrolyzer interfacedto the DESI source; FIG. 6(B) is a diagrammatical view of the on-probepyrolyzer.

FIG. 7(A) is a diagrammatical view of the on-probe pyrolysis (220° C.,11 s) DESI-mass spectrum of Angiotensin II (inset: before pyrolysisDESI-mass spectrum); site-specific cleavage is induced at the C-terminusof aspartic acid. Ions at m/z 1028 and 1011 are the result ofdehydration and deamination reactions, and FIG. 7(B) is a diagrammaticalview of the on-probe pyrolysis DESI-tandem mass spectrum of the ion atm/z 931.

FIGS. 8(A-C) are diagrammatical views of (a) the on-probe pyrolysisDESI-mass spectrum of the VIP peptide showing site-specific cleavages atthe two aspartic acids amino acids (inset: before pyrolysis DESI-massspectrum); and the on-probe pyrolysis DESI-tandem mass spectrum ofpyrolytic product at (b) m/z 1086 and (c) m/z 553.

FIG. 9(A) is a diagrammatical view of the on-probe pyrolysis DESI-massspectrum of lysozyme (inset: before pyrolysis DESI-mass spectrum); andFIG. 9(B) a diagrammatical view of the on-probe pyrolysis DESI-tandemmass spectrum of the ion at m/z 1201.

FIG. 10 is the on-probe pyrolysis DESI-mass spectrum of the proteinRNase A (inset: before pyrolysis DESI-mass spectrum).

FIG. 11(A) is the DESI-mass spectrum of PEG 2000 before pyrolysis, andFIG. 11(B) is the on-probe pyrolysis (250° C., 30 min) DESI-massspectrum of PEG 2000 (inset: zoomed mass spectrum in the range 840-970Da).

FIG. 12(A) is and ESI-MS spectrum of pyrolysis products of the VSV-Gpeptide showing site specific cleavage at the aspartic acid site; andFIG. 12(B) is an MS² spectrum of the specific pyrolysis products of theVIP (1-12) peptide, confirming its sequence.

FIG. 13 is a full scan mass spectrum of pyrolysis products of peptideACTH (1-10); the cleavage product at E with m/z 702 was not observed.

FIG. 14 is an ESI full scan mass spectrum of pyrolysis products of theprotein lysozyme.

FIGS. 15 (A-E) are charts of the MS/MS spectra of ions m/z 586.5, 605.6,828.6, 1327.6, and 1434.6, respectively.

FIG. 16 is a chart of the observed peptide fragments according to thecleavage rules after pyrolysis of protein lysozyme.

FIG. 17 is a chart of the disulfide bond sites in lysozyme.

FIG. 18(A) is a chart of the ESI full scan of peptide Antioxidant Aafter pyrolysis and FIG. 18(B) the MS/MS spectrum of m/z 505, theC-cleavage product.

FIG. 19(A) is a chart of the ESI full scan of peptide Somatostatin 14after pyrolysis and FIGS. 19(B) and 19(C) the MS/MS spectrum of m/z1535.6 and 1575.6, the C-cleavage products, respectively.

FIGS. 20A-C show the Mascot search results of lysozyme.

FIGS. 21 (A)-(C) show the ESI full scan mass spectrum of the proteininsulin after pyrolysis and the MS/MS mass spectra of m/z 566 and 778.

FIG. 22 shows the Mascot search result of insulin using the MS/MS dataof ions at m/z 778 and 566.

FIGS. 23A-F show the ESI full scan of protein α-lactalbumin (bovinemilk) after pyrolysis and the MS/MS spectra.

FIGS. 24 (A)-(C) show the ESI full scan mass spectrum of the proteinalbumin (chicken) after pyrolysis and the corresponding MS/MS massspectra.

FIG. 25(A) shows the ESI full scan mass spectrum of the proteincytochrome C (horse heart) after pyrolysis and FIG. 25 (B) shows thecorresponding MS/MS mass spectra.

FIG. 26 shows the data file of ion m/z 1263 showing the identificationof cytochrome C.

FIG. 27 shows the chromatogram and the Sequest search result ofpyrolyzed lysozyme.

FIGS. 28 (A)-(C) show chromatograph (A) and full MS scans (B)-(C) attime 22,58 min and 39.17 min for protein alactalbumin.

FIGS. 29 (A)-(B) show the full ESI mass spectrum scan of pyrolyzedsamples of E. coli and S. Aureus via direct infusion ESI-MS.

FIGS. 30 (A)-(B) show the on-probe DESI-MS full scan of pyrolyzedbacteria whole cells Salmonella typhimutium (A), and Pseudomonasaureginosa (B).

FIGS. 31 (A)-(C) show chromatograph (A) and full scan mass spectra forbacteria E. coli at retention time 25.34 min (B) and 25.85 min (C).FIGS. 32 (A)-(C) show chromatograph (A) and full scan mass spectra forbacteria S. aureus at retention time 13.43 min (B) and 33.32 min (C).

FIGS. 32 (A)-(C) show chromatograph (A) and full scan mass spectra forbacteria S. aureus at retention time 13.43 min (B) and 33.32 min (C).

FIG. 33 shows an HPLC chromatogram of cytochrome c and a chart ofSequest results.

FIG. 34(A) is an HPLC chromatogram of pyrolyzed lysozyme and FIG. 34(B)is a chart of the Sequest search result gained via HPLC-MS.

FIG. 35 is a chart of Sequest results gained via HPLC-MS.

FIGS. 36(A) and (C) are HPLC chromatograms of alpha-lactalbumin andFIGS. 36(B) and 36(D) are charts of the full MS scans at time 22.58 minand 39.17 min respectively, showing the peptides eluting out of thecolumn.

FIGS. 37(A) and 37(B) are charts of the full ESI scan of pyrolyzed wholecells of E. coli and S. aureus, respectively.

FIGS. 38(A) and 38(B) are charts of the on-probe DESI-MS full scan ofpyrolyzed whole cells of S. typhimutium and P. aureginosa, respectively.

FIG. 39(A) is an HPLC chromatogram of pyrolyzed E. coli, FIGS. 39(B) and39(C) are charts of the full MS scans at 25.34 and 25.85, respectively,and FIG. 39(D) is a chart of the Sequest search in a bacteria databaseshowing the one of its possible origins is E. coli.

FIG. 40(A) is an HPLC chromatogram of pyrolyzed S. aureus, FIGS. 40(B)and 40(C) are charts of the full MS scans at 13.43 and 33.23,respectively, and FIG. 40(D) is a chart of the Sequest search in abacteria database showing the one of its possible origins is S. aureus.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1Pyrolysis-Induced Cleavage at Aspartic Acid Residue in Peptides andProteins

Chemicals.

Peptides used were: (A) Angiotensin II, human, DRVYIHPF (SEQ ID NO. 1);(B) VIP (1-12) peptide, HSDAVFTDNYTR (SEQ ID NO. 2); and (C) VSV-Gpeptide, YTDIEMNRLGK (SEQ ID NO. 3) (all from AnaSpec, San Jose,Calif.). Lysozyme protein (from Sigma-Aldrich, St. Louis, Mo.) was usedwithout further purification. All solvents used for sample preparationand MS measurements were HPLC grade (Burdick & Jackson, Muskegon,Mich.), and the formic acid (96%) was ACS Reagent grade (Aldrich, St.Louis, Mo.).

Pyrolyzer Design and Pyrolysis Procedure.

Approximately a 1 mg solid sample of peptide or protein was pyrolyzedunder ambient conditions. Samples were placed in a glass tube (length 31mm and internal diameter 4 mm; Agilent, Santa Clara, Calif., Part#5180-0841) and heated using a resistance heating wire (Omega, Stamford,Conn., nickel-chromium wire, part # NI60-015-50, length 20 cm) enwoundaround the tube, powered by 13 V alternating current (AC). Temperaturewas measured in situ using a thermocouple probe (model HH12A, OmegaCompany, Stamford, Conn.) reaching down the bottom of the glass tube.The sample was heated for 10 s under atmospheric condition to a finaltemperature of 220° C. The nonvolatile pyrolysis residue was collectedby washing/extracting the inside of the tube with 1 mL of a 50/50 (v/v)methanol-water solution with 0.1% formic acid (FA).

Mass Spectrometry.

The extracted solution of pyrolysis products was directly analyzed usinga quadrupole ion-trap MS (LCQ classic, Finnigan, San Jose, Calif.)equipped with a nano-Electrospray Ionization (nano-ESI) source byinfusing it into the mass spectrometer at a flow rate of 3 μL/min via a250-μL syringe. Tandem MS (MS/MS) was conducted with the followingparameters: activation q of 0.250; isolation width was 1 amu, and thepercentage relative collision energy was in the range of 25-40% and wasadjusted such that the relative abundance of the precursor ion in theproduct ion spectrum was approximately 30-50% relative intensity.

MALDI-MS experiments were performed using a MALDITime-of-Flight MS(Voyager DE-PRO, Applied Biosystems, Foster City, Calif.) instrumentequipped with a N₂ laser and operated in the reflectron mode. The matrixR-cyano-4-hydroxy-cinnamic acid (CHCA) (Aldrich) was used for allmeasurements and was prepared by dissolving 10 mg of CHCA in a 1 mLsolution of 1:1 acetonitrile/water with 0.1% trifluoroacetic acid (TFA)(Pierce Chemical Co., Rockford, Ill.). The extracted solution ofpyrolysis products was directly mixed with the matrix at differentvolume ratios and air-dried onto a MALDI plate.

Results and Discussion

Analysis of Nonvolatile Pyrolysis Products of Peptides.

Three peptides containing aspartic acid were pyrolyzed, theirnonvolatile products were analyzed by ESI-MS, and their amino acidsequences were confirmed by tandem MS: (A) Angiotensin II, human,DRVYIHPF (SEQ ID NO. 1); (B) VIP (1-12) peptide, HSDAVFTDNYTR (SEQ IDNO. 2); and (C) VSV-G peptide, YTDIEMNRLGK (SEQ ID NO. 3). FIGS. 1A-Cshow the full scan mass spectra of peptide A before and after pyrolysisat different temperatures.

For the peptides tested at pyrolysis temperatures of 200° C. and lower(data not shown), no significant pyrolysis fragments were detected. Onthe other extreme, at a pyrolysis temperatures of 290° C. and higher(data not shown), extensive fragmentation products were observed, mostlikely due to peptide carbonization. At pyrolysis temperatures between220 and 250° C., the pyrolysis fragment for peptide A due to C-terminalcleavage at aspartic acid was detected at m/z 931.6. Tandem MS (MS/MS)of this ion (FIG. 2) confirmed the sequence RVYIHPF, the product of aC-terminus cleavage at the aspartic acid residue of peptide A. Otherpeaks present in the spectrum resulted from consecutive loss of water,observed at m/z 1028.5 (C-terminus oxazolone formation) (Zhang, S.;Basile, F. Investigation of Non-Volatile Pyrolysis Products of ProteinsUsing Electrospray Ionization Multi-step Tandem Mass Spectrometry. 54thASMS Conference, 2006), and loss of ammonia, observed at m/z 1011.5(from arginine).

Site-specific pyrolysis-induced cleavage was also observed for peptideB, which contains two aspartic acid residues (ESI-mass spectrum shown inFIG. 3A). After pyrolysis at 220° C., two nonvolatile peptide productswere observed at m/z 1086.5 (AVFTDNYTR) (SEQ ID NO. 4) and m/z 553.6(NYTR) (SEQ ID NO. 5), corresponding to cleavages at each of the twoaspartic acid C-terminus sites. Amino acid sequences of these pyrolysisproducts were confirmed by MS/MS measurements (shown in FIGS. 4A and4B). Also, possible peptide oxidation products were observed at m/z 1521for peptide B and at m/z 1435 for peptide C, and their structures arecurrently being investigated. Similar results were observed for peptideC (FIG. 3B).

These results demonstrate that pyrolysis at temperatures between 220 and250° C. favors cleavage in peptides at the C-terminus of aspartic acid.Peptide fragmentation at the C-terminus of aspartic acid is believed toproceed via the formation of a five-member cyclic anhydride followed byhydrolysis, because pyrolysis is performed in air and at atmosphericpressure (Scheme 1) (Inglis, S. A. Cleavage at aspartic acid. MethodsEnzymol. 1983, 91, 324-332).

The overall susceptibility of the aspartic acid group to internalcleavage may stem from the fact that the {circumflex over (α)}-carboxylgroup (i.e., side-chain carboxyl group) acts as a proton donor and itshydroxyloxygen as a nucleophile toward the adjacent carbonyl carbons inthe peptide bond. Reaction path “a” in Scheme 1 leads to the formationof a five-member ring, while path “b” forms a six-member ring species.Hydrolysis of these cyclic intermediates results in C- or N-terminuscleavages of the aspartic acid residue, respectively. The six-memberring molecule leading to the N-terminus cleavage is expected to bethermodynamically more stable than the five-member cyclic anhydridemolecule (Loudon, G. M. Organic Chemistry; Addison-Wesley PublishingCo.: Massachusetts, 1983). However, only C-terminus cleavage productshave been detected under pyrolysis conditions, and these would resultfrom the formation of the five-member ring species. Hence, it ishypothesized that the reaction path “a” leading to the pyrolysis-inducedC-terminus cleavage is kinetically favored rather than thermodynamicallycontrolled.

Analysis of Nonvolatile Pyrolysis Products of Lysozyme.

The potential of this methodology to digest intact proteins to smallerpeptides for subsequent MS/MS analyses was further tested. MALDI-MSanalysis of the nonvolatile pyrolysis products of the protein lysozymeresulted in a series of strong signals in the mass range of 500-2500 u,indicating that complete degradation or carbonization of the proteindoes not occur at 220° C. (FIG. 5A). Moreover, the ion at m/z 1201.6observed in the MALDI-mass spectrum matches one of the expected productscorresponding to the cleavage at the C-terminus of aspartic acid inlysozyme, the peptide (D) VQAWRGCRL (SEQ ID NO. 6).

Analysis of this pyrolysis digestion product by ESI-MS/MS (MS/MS of m/z1201.6) yielded sequence information confirming the peptide amino acidsequence (FIG. 5B). Moreover, fragment ion data were used for successfulprotein identification via database search (Mascot score 52; thresholdscore for significant homology was 43; Matrix Science, UK). Hence, theobserved pyrolysis digestion product at m/z 1201.6 corresponds to theC-terminal peptide in the lysozyme protein sequence, confirming that thepyrolysis product is derived from the protein and that sequenceinformation is conserved. We are currently investigating factorsaffecting sequence coverage and the structure of additional pyrolysisproducts observed (e.g., dehydration, deamination, and oxidationproducts), the effect of neighboring amino acids on cleavage, and theability of the method to cleave at other aspartic acid residues, thatis, other than those near the C-terminal of the protein sequence.

Conclusion.

The ability of pyrolysis-based digestion methods to producesequence-specific biomarkers has been demonstrated for peptides and theprotein lysozyme. This approach offers the possibility of developingrapid and field-portable proteomic-based methods to detect and identifybiological samples, for example, protein toxins and/or pathogenicbacteria (e.g., Bacillus anthracis). In this particular application,protein sequence coverage is not a requirement, but, rather, thereproducibility and simplicity of the pyrolysis method is used togenerate biological-specific biomarkers.

Example 2 Pyrolysis Device and Procedures

A diagram of the on-probe pyrolyzer interfaced to the DESI source isshown in FIG. 6A. A homebuilt DESI source was interfaced with aquadrupole ion trap MS (LCQ Classic, Thermo Electron, San Jose, Calif.)and was operated in the positive ion mode. The on-probe pyrolyzerconsisted of a membrane heater (Model #HM6815, Minco, Minneapolis,Minn.) placed underneath a removable glass slide held tightly togetherwith a clamp (FIG. 6B). The sample to be pyrolyzed was placed directlyon the center of the glass slide. The membrane heater was powered byalternating current (AC) from a transformer (Model #3PN116C, SuperiorElectric, Farmington, Conn.) and heating and final pyrolysis temperaturewere controlled by adjusting the voltage of the transformer and theheating time. For our current setup, a voltage of 20 V applied for 11 sresulted in a final pyrolysis temperature of 220° C. These values forpyrolysis temperature and time were used for all biological samplesanalyzed in this study. The glass slide surface temperature was measuredin situ using a thermocouple probe (Model #HH12A, Omega Company,Stamford, Conn.) placed in direct contact. After sample pyrolysis, theprobe was cooled to room temperature (<5 min) and the DESI-MS analysiscarried out. This setup is amenable to conducting pyrolysis in eitherthe off-line or on-line mode with the DESI source, that is, a sampleplaced on a slide can be pyrolyzed in a furnace under controlledatmospheric conditions and later analyzed by DESI-MS. However, allmeasurements in this report were performed in the on-line configuration(FIG. 6A).

Several model samples were tested with this new on-probe pyrolyzerDESI-MS instrument. Peptides analyzed included Angiotensin II-human, ofsequence DRVYIHPF (SEQ ID NO. 1), and the peptide VIP (1-12), ofsequence HSDAVFTDNYTR (SEQ ID NO. 2) (both from AnaSpec, San Jose,Calif.). The proteins used were lysozyme and RNase A, and the syntheticpolymer used was poly(ethylene glycol) (PEG 2000) (all fromSigma-Aldrich, St. Louis, Mo.). Methanol, water (from Burdick & Jackson,Muskegon) and tetrahydrofuran (THF, from EMD Chemicals, San Diego,Calif.) were used for sample preparation and MS measurements (all HPLCgrade). About a 1 mg sample of the peptides was dissolved in 200 μL ofmethanol, and the entire solution air-dried on a glass slide (covering asurface area approx. 6 cm², ˜0.1 mg sample/cm²) and placed on theon-probe pyrolyzer. Lysozyme and RNase A were prepared in a similarfashion, but dissolved in water. For poly(ethylene glycol), about 10 mgof PEG 2000 was dissolved in 1 mL of THF, air-dried on a glass slide (˜1mg/cm²), placed on the on-probe pyrolyzer and heated to a finaltemperature of 250° C. for 30 min.

DESI and Mass Spectrometry Parameters

The DESI source was operated with a high voltage of 6 kV applied to thespraying solvent. The spraying solvent consisting of 50% methanol inwater (v/v) was delivered at a flow rate of 7 μL/min via a syringe pump.All mass spectra were collected in spectral average mode. The pressureof the DESI nebulizer gas (N₂) was set as 250 psi.

Tandem MS (MS/MS) measurements were conducted with the followingparameters: activation q of 0.250; isolation width was 1 amu and thepercentage relative collision energy was in the range of 25-40%, and wasadjusted to get a precursor ion peak of 25% relative intensity or less(when possible).

Results and Discussion

The utility and versatility of the DESI source interfaced with theon-probe pyrolyzer for the analysis of non-volatile pyrolysis productswere demonstrated with several model compounds that included peptides,proteins and a synthetic polymer.

Example 3 On-Probe Pyrolysis DESI-MS Analysis of Biomolecules

As described in Example 1, the site-specific pyrolysis-induced cleavageat the amino acid aspartic acid (letter symbol “D”) in both peptides andproteins has been achieved by heating samples to a temperature of220-250° C. for 10 s under atmospheric pressure conditions. Peptides andproteins in this previous study were pyrolyzed in an open-ended tubefurnace, extracted with a suitable solvent and analyzed by ESI-MS andMS/MS to characterize and identify non-volatile pyrolysis cleavageproducts. In this Example, the same samples were pyrolyzed on-probe andproducts were analyzed in situ by DESI-MS, bypassing the sampleextraction, transfer, and ESIinfusion steps. In the ESI-MS study and theDESI-MS study here described, pyrolysis of peptides and proteins above300° C. produced complete charring of the polypeptide backbone.

Pyrolysis induced site-specific cleavage at aspartic acid has wasobserved mostly at low temperature pyrolysis. However, this pyrolysiscleavage reaction is not exclusive in biomolecules as other pyrolysisfragments have been detected and the system here described is presentlybeing used to further characterize the structure and nature of thesepyrolysis fragments.

FIGS. 7A and 7B illustrate the DESI-mass spectra before and after onprobe pyrolysis of the peptide Angiotensin II, along with the tandemmass spectrum of the pyrolytic product at m/z 931. The DESI-massspectrum of the non-volatile products also shows the formation of adehydration product at m/z 1028.2, a possible oxidation product at m/z1124.1 (of yet unknown structure) and the product of the pyrolysisinduced site-specific cleavage at aspartic acid at m/z 931.2 (theD-cleavage pyrolysis peptide product). Tandem MS data of the ion at m/z931 confirms that sequence-specific information is preserved after lowtemperature pyrolysis of peptides.

The above measurement demonstrates the simplicity and speed of analysisof pyrolysis residues with the on-probe pyrolyzer coupled to a DESI-MSsystem. No solvents were required for residue extraction andsolubilization, assuring the analysis of the entire pyrolysis productmixture (i.e., the nonvolatile fraction, vide infra). However, it isreassuring to note that all products detected in the on-probe pyrolysisDESI-MS analysis in FIGS. 8A-C were also observed in the open-ended tubefurnace pyrolysis and ESI-MS analysis, which required sample extractionand solubilization. It is important to note that lower MW products likediketopiperazines (DKP) known to be generated under Curie-point andatmospheric pyrolytic conditions were only observed in the analysis ofthe Angiotensin II peptide (signal at m/z 263 corresponding to the(M+H)+DKP of VY). This may be due to several factors: first, volatileDKP products may have been lost during the pyrolysis process since theon-probe pyrolyzer is operated at atmospheric pressure. Second, earlywork on the formation of DKP from dipeptides (D. Gross, G. Grodsky, J.Am. Chem. Soc. 77 (1955) 1678-1680; H. J. Svec, G. A. Junk, J. Am. Chem.Soc. 86 (1964) 2278-2282) found that only a small percentage (˜7%) ofthe original dipeptide was converted to DKP at 215° C. And finally,ionization suppression of the DKP (M+H)+signals within the desorbed DESIdroplets may take place, especially if analyzing a complex mixture ofpyrolytic products with dissimilar droplet surface activities or DKPs inmixtures with peptides containing highly basic groups (i.e., arginine),as it is the case here.

FIGS. 9A and 9B illustrate the on-probe pyrolysis and DESI-MS analysisof another peptide, VIP (1-12) peptide, which contains two aspartic acidresidues. Specifically, the on-probe pyrolysis DESI-mass spectrum (FIG.8A) is characterized by the ions at m/z 553.6 and 1086.3, whichcorrespond to the expected products due to site-specific cleavages atthe two aspartic acid residues (D-cleavage pyrolysis). This D-cleavagepyrolysis is believed to proceed via a similar mechanism as in thesolution phase reaction, that is, the formation of a five-member cyclicanhydride followed by hydrolysis. Similar results were also obtained inthe open-ended tube furnace (at atmospheric pressure conditions) andsolvent extraction ESI-MS analysis of the pyrolysis residues. Other ionsobserved at m/z's 1068 and 1050 result from consecutive losses of waterand ammonia (from arginine) from the pyrolysis fragment at m/z 1086.3,and these ions were also observed in the off-line pyrolysis andextraction ESI-MS measurements. FIGS. 8B and 8C show the on-probeDESI-tandem mass spectra of the pyrolysis products at m/z 1086 and 553,confirming their sequences and the site-specificity of the pyrolysiscleavage at aspartic acid. Also, the on-probe pyrolysis DESI-MSinstrument was used to analyze the non-volatile pyrolysis products ofthe proteins lysozyme (MW 14.3 kDa) and RNase A (MW 13.7 kDa). FIGS. 9Aand 9B show the DESI-mass spectra of lysozyme before and after pyrolysisand the DESI-tandem mass spectrum for the ion at m/z 1201. This ioncorresponds to the protein C-terminus peptide due to D-cleavagepyrolysis as confirmed by the DESI-tandem mass spectral data in FIG. 9B.In previous work, it was successfully shown that this sequenceinformation can be used to identify the protein via a proteomic-basedapproach and database search (e.g., MASCOT, Matrix Science Ltd., London,UK). FIG. 10 illustrates the on-probe pyrolysis DESI-MS analysis of theprotein RNase A with the detection of several prominent pyrolysisproducts observed at m/z's 437.3, 789.5, 916.4, 1047.5 and 1212.4;however, none of the main signals observed match expected productsresulting from D-cleavage pyrolysis. In previous investigations and inthis study, the D-cleavage pyrolysis peptide product was derived fromthe C-terminus of the protein sequence, and not from cleavages ofinternal D groups.

On-Probe DESI-MS Analysis of Poly(Ethylene Glycol)

Poly(ethylene glycol) with an average molecular weight of 2000 g/mol(PEG 2000) was used to test the ability of the on-probe pyrolyzerDESI-MS instrument to study thermal degradation processes in syntheticpolymers. FIGS. 11A and 11B show the DESI-mass spectra of the PEG 2000before and after on probe pyrolysis at 250° C. for 30 min. The DESI-massspectrum of untreated PEG 2000 (FIG. 6A) shows a distribution of singlycharged ions near m/z 2000 as their (M+Na)⁺ ions (monomer unit Dm=44 u)denoted in the spectrum as the P⁺-series. A doubly charged P²⁺-series isalso observed near m/z 1000 (monomer unit Dm=22 u) and is composed ofboth (M+2Na)²⁺ and (M+Na⁺ K)²⁺ ions. On the other hand, the on-probepyrolyzed DESI-mass spectrum of PEG 2000 (FIG. 11B and inset) isstrikingly different, with the P⁺ series shifted to an average molecularweight near m/z 1000, while the P²⁺ and P³⁺ were not detected. Carefulinspection of this mass spectrum (FIG. 11B inset) reveals the presenceof several series of poly(ethylene glycol) with different end groups,and these are labeled using nomenclature coined by Voorhees et al.(Voorhees, K. J., Baugh, S. F., Stevensen, D. N. J. Anal. Appl. Pyrol.30 (1994) 47-57)). The spectrum in FIG. 11B is dominated by theunmodified hydroxylpoly(ethylene)glycol series (labeled N in thespectrum), methyl ether series (A), aldehyde series (C) and the ethylether series (D). Less dominant, but present, are the vinyl ether series(B), the methyl ether/aldehyde series (E) and the methyl-vinyl etherseries (C0). These results are in direct agreement with previousMALDI-MS studies (Lattimer, R. P. J. Anal. Appl. Pyrol. 56 (2000) 61-78)of the pyrolyzate residues of poly(ethylene glycol), proving that theon-probe pyrolysis DESI-MS technique described in this report yieldscomparable results. Moreover, the on-probe pyrolysis DESI-MS approachdoes not require matrix compounds, decreasing sample preparation timeand avoiding matrix-sample adducts that can add to the chemical noise inthe mass spectrum. Also, in this Example, no cationizing agent was addedto either the polymer sample or the DESI solvent, and we believe thesource of the Na⁺ ions to be the glass slide and/or from trace amountscontained in the DESI solvent.

Conclusion

An on-probe pyrolyzer interfaced with desorption electrospray ionization(DESI) mass spectrometry and tube pyrolysis with sample extraction weresuccessfully demonstrated to induce site specific cleavage at asparticacid in biological samples. These results are in agreement with analysesof non-volatile pyrolysis products performed either by ESI-MS orMALDI-MS, which were pyrolyzed off-line and required sample extractionand solubilization. For biological samples and using the on-probepyrolyzer DESI-MS system, it has here been demonstrated that pyrolysisresidues of peptides and the protein lysozyme retain sequenceinformation useful for proteomic-based protein identification. Moreover,these results demonstrate that atmospheric pressure pyrolysis can inducea variety of products that include site-specific cleavages at asparticacid, dehydration reactions in peptides and proteins, and otherproducts. For the analysis of poly(ethylene glycol), the on-probepyrolysis DESI-MS system yielded data and information equivalent toprevious MALDI-MS analysis, where the use of a matrix compound andcationizing agent were required. Quantitative to semi-quantitativeanalysis with DESI-MS is feasible, although quantitation of pyrolysisproducts was not addressed in this work. Overall, results from this workhave demonstrated clear advantages of combining an on-probe pyrolyzerwith a DESI source that include: minimum sample preparation, no sampleextraction or transfer after pyrolysis, atmospheric pressure pyrolysis,rapid and atmospheric pressure detection by DESIMS, the ability forsample archival (samples on slides), and tandem-MS (if using amultistage-MS system).

Example 4 Site Specific Cleavage at Aspartic Acid Residue

This example concerns, inter alia, an automation of the samplepreparation step for bottom-up proteomic analyses for microorganisms(i.e., biodetection) using a rapid reagentless approach forsite-specific cleavage of peptides and proteins based on pyrolysis.

Experimental

The pyrolysis experiments were conducted using the device describedpreviously in Example 1.

Chemicals

Peptides used were: 1) Angiotensin II, human DRVYIHPF (SEQ ID NO. 1); 2)VIP (1-12) peptide, HSDAVFTDNYTR (SEQ ID NO. 2); 3) VSV-G Peptide,YTDIEMNRLGK(SEQ ID NO. 3); 4) ACTH (1-10), SYSMEHFRWG (SEQ ID NO. 7); 5)Antioxidant peptide A, PHCKRM (SEQ ID NO. 8); 6) Somatostatin 14,AGCKNFFWKTFTSC (SEQ ID NO. 9) (all from AnaSpec, San Jose, Calif.). Theproteins: insulin (bovine pancreas, MW 5.7K), α-lactalbumin (bovinemilk, MW 14K), lysozyme (chicken egg white, MW 14.3K), albumin (chickegg white, MW 42.8K), cytochrome C (horse heart, MW 11.7K) and MALDImatrix: sinapinic acid (SA) and α-cyano-4-hydroxycinnamic acid (CHCA)were all bought from Sigma and used without further purification. Allsolvents (water, methanol, acteonitrile (ACN)) used for samplepreparation and MS measurements were HPLC grade (Burdick & Jackson,Muskegon, Mich.) and the formic acid (FA 96%) was ACS reagent grade(Aldrich, St. Louis, Mo.).

Bacteria

Escherichia coli (E. coli), Staphylococcus aureus (SA), Pseudomonasaureginosa (PA), Salmonella typhimutium (ST), were purchased fromAmerican Type Culture Collection (ATCC, Manassas, Va.). Bacteria weregrown under optimum laboratory conditions using tryptic soy agar (TSA;BD Science, Sparks, MD) at 37° C. for 12-15 h. Bacterial cells weretransferred to 1 mL of water using a sterile tungsten loop inoculator.The cells were vortexed for 10 seconds and centrifuged for 5 minutes at12,000 rpm to remove the media. The supernatant was removed and thepellet was re-suspended in 1 mL of water and the washing step wasrepeated for 3 times. After three washing steps the cells were dried byvacuum centrifuge before being pyrolyzed.

Mass Spectrometry and HPLC

The extracted solution of pyrolysis products was directly analyzed byHPLC separation step followed by online quadrupole io-trap MS (LCQclassic, Thermo Finnigan, San Jose, Calif.) equipped with anano-electrospray ionization (nano-ESI) source.

For direct ESI analysis, sample was infused into the mass spectrometerat a flow rate of 3 μL/min via a 250-1 μL syringe (Harvard Apparatus,Holliston, Mass.) via a syringe pump. The mass spectra were collectedusing the LCQ Tune Plus software (Thermo Finnigan, San Jose, Calif.).Tandem MS (MS/MS) was conducted with the following parameters:activation q of 0.250; isolations width was 1 amu and the percentagerelative collision energy was in the range of 25-40%, and was adjustedsuch that the relative abundance of the precursor ion in the product ionspectrum was approximately 30-50% relative intensity.

For HPLC-ESI-MS analyses, peptide mixtures from pyrolysis products wereseparated using an Ultimate/Switchos HPLC system (LC Packing, Sunnyvale,Calif.). A 1 μL aliquot of a peptide mixture solution was loaded onto aseparation column (PepMap, C18, 150 mm×75 μm, 100 Å, Dionex, Sunnyvale,Calif.), which had been equilibrated with 95% A (Solvent A: 20% ACNcontaining 0.1% FA, Solvent B: 100% CAN containing 0.1% FA). Peptideswere separated using the following gradient at a flow rate of 300mL/min: 0-10 min, 5% B; 10-60 min, 5%-60% B; 60-90 min, 100% B; 90-130min, 100% A. The HPLC column eluate was connected to thenano-electrospray ionization (nESI) source of the LCQ. Tandem MSexperiments with the ion trap MS were performed by acquiring a full-scanmass spectrum between m/z 300 and 2000 followed by three data dependentproduct ion mass-spectral scans of the most intense precursor ions(a.k.a “big-three” scan). The collision energy for dissociation was setat 30% with a 50 ms activation time. The dynamic exclusion feature ofthe Xcalibur software was enabled with a repeat count of 2, a repeatduration of 0.5 min, and exclusion duration of 2 min.

MALDI-MS experiments were performed using a Voyager DE-PRO (AppliedBiosystems, Foster City, Calif.) instrument equipped with a N₂ laser andoperated in the reflection mode. The matrix α-cyano-4-hydroxy-cinnamicacid (CHCA) was used for all measurements and was prepared by dissolving10 mg of CHCA in a 1 mL solution of 1:1 acetonitrile/water with 0.1%trifluoroacetic acid (TFA) (Pierce Chemical Company, Rockford, Ill.).The extracted solution of pyrolysis products was directly mixed with thematrix at different volume ratios and air-dried onto a MALDI plate.

Results and Discussions 1. Pyrolysis Induced Cleavage at Aspartic Acid(D-Cleavage)

Several model peptides containing aspartic acid were pyrolyzed, theirnon-volitile products analyzed by ESI-MS, and their amino acid sequencesconfirmed by tandem MS: 1, Angiotensin II, human, DRVY1HPF (SEQ ID NO.1); 2, VIP (1-12) peptide, HSDAVFTDNYTR (SEQ ID NO. 2); 3, VSV-GPeptide, YTDIEMNRLGK (SEQ ID NO. 3); 4, ACTH (1-10): SYSMEHFRWG (SEQ IDNO. 7). Angiotensin II was pyrolyzed at different temperatures to probethe pyrolysis temperature's effect on the distribution of pyrolyticproducts. Angiotensin II was tested at pyrolysis temperatures of 200,200, 250 and 290° C. The ESI-mass spectra of the pyrolytic productsunder various temperatures are shown in FIGS. 1A-C.

For the peptides tested at pyrolysis temperature of 200° C. The ESI-massspectra of the pyroltic products under various temperatures are shown inFIGS. 1A-C.

For the peptides tested at pyrolysis temperatures of 200° C. and lower,no significant pyrolysis fragments were detected. At the other extreme,pyrolysis temperatures of 290° C. and higher, yielded extensivefragmentation products which were most likely due to peptidecarbonization. At pyrolysis temperatures between 220° C. and 250° C.,several products are detected and correspond to the pyrolysis fragmentdue to C-terminal cleavage at aspartic acid (detected at m/z 931.6).Tandem MS (MS/MS) of this ion (FIG. 2) confirmed the sequence RVYIHPF(SEQ ID NO. 10), the product of a C-terminus cleavage at the asparticacid residue of angiotensin II. Other peaks present in the spectrum wereobserved at m/z 1028.5 (18 amu less than parent ion) and at m/z 1011.5(17 amu less), which are believed to be a water loss (from theC-terminus) and ammonia loss (from arginine) respectively.

The same experiments were carried out on VIP (1-12) peptide, of sequenceHSDAVFTDNYTR (SEQ ID NO. 2). Site specific pyrolysis-induced cleavagewas also observed for this peptide, which contains two aspartic acidresidues (ESI-mass spectrum shown in FIGS. 3A and 3B). After pyrolysisat 220° C., non-volatile peptide products are observed with the ions atm/z 1086.5 (AVFTDNYTR) (SEQ ID NO. 4) and m/z 553.6 (NYTR) (SEQ ID NO.5), corresponding to cleavages at each of the two aspartic acidC-terminus sites. Amino acid sequences of these pyrolysis products wereconfirmed by MS/MS measurements (shown in FIGS. 4A and 4B). Also,another peptide reaction products was observed at m/z 1521 for VIP(1-12) peptide corresponding to the addition of trifluoroacetic acid(TFA, used to acidify solutions) to the N-terminus and at m/z 1435 forVSV-G Peptide (FIGS. 12A and 12B).

The same experiments were carried out for VSV-G Peptide, YTDIEMNRLGK,which contains the amino acids D and E (glutamic acid). The ESI fullscan spectrum of pyrolytic products is shown in FIGS. 5A and 5B. Thesignal at m/z 960.7 corresponds to the sequence of 1EMNRLGK (SEQ ID NO.11), a product corresponding to cleavage at D. However, a new peak atm/z 718.4 was also observed MS² experiment of this peak shows itssequence is MNRLGK (SEQ ID NO. 12), a pyrolytic product corresponding tocleavage at the C-terminus of E. Considering the similarity between Dand E where both of them have a carboxylic acid in their branch chains,more experiments were don to test where E is a possible cleavage site,hence other peptides contains E but without D were tested. Peptide ACTH(1-10): SYSMEHFRWG (SEQ ID NO. 7) was tested. The full scan massspectrum of its pyrolyzed products is shown in FIG. 13, however theexpected cleavage product of E of m/z 702 was not observed.

These results demonstrate that pyrolysis at temperatures between 220° C.and 250° C. favors cleavage in peptides at the C-terminus of D. Peptidefragmentation at the C-terminus of D is believed to proceed via theformation of a five-member cyclic anhydride followed by hydrolysis,since pyrolysis is performed in air and at atmospheric pressure.

Example 5 Pyrolysis Induced Cleavage at Cysteine (C-Cleavage)

The ability of the pyrolysis-based digestion method to producesequence-specific biomarkers has been demonstrated for peptides and theprotein lysozyme. However, only the fragment near to the C-terminus ofthe protein sequence has been identified, namely m/z 1201 correspondingto the D cleavage. In order to identify other prominent peaks in FIGS.8A-C such as 828, 1328 etc., tandem MS measurements were conducted onthem.

The ESI full scan spectrum of pyrolyzed lysozyme at 220° C. is shown inFIG. 14 and the MS/MS spectra of m/z 586.5, 605.6, 828.6, 1327.6, 1434.5are shown in FIGS. 15A-E, respectively.

Careful inspection of this serial of MS/MS mass spectra (FIGS. 15A-E)reveals that pyrolysis indeed induced the cleavage of the proteinlysozyme mot only at the C-terminus of D, but also induced cleavages atthe N-terminus of cysteine residues, with the added −1 Da massmodification at the amino acid to the C terminal side of the cysteine.The structure of the −1 mass modification is under further investigationand we believe it to be an amide (R—CO—NH₂) replacing the original —OHgroup at the C-terminus of a peptide. This result shows that pyrolysisis a very promising protein digestion approach, as it can generate smallpeptide fragments (which are desired by mass spectrometry measurements)by site-specifically cleaving at two different amino acid sites. FIG. 16shows the observed peptide fragments according to the (revised)pyrolysis DC-cleavage rules and it can be seen that the majority of theprominent peaks are assigned.

When both of the two-site cleavages considered, very good sequencecoverage (42%) of lysozyme is obtained (FIG. 16). Worth to note thatdisulfide bonds connecting two cysteine residues in a polypeptide chainmust be broken during the pyrolysis of lysozyme as well, when we take alook at the disulfide bond sites in lysozyme (FIG. 17). The disulfidebonds have to be broken to generate the observed peptide fragments ofsequences

*C (SEQ ID NO. 13) (MH⁺ 605.7), D

*C (SEQ ID NO. 14) (MH⁺ 1327.8), D

*C (SEQ ID NO. 15) (MH⁺ 1434.6), D

*C (SEQ ID NO. 16) (MH⁺ 956.8), D

(SEQ ID NO. 17) (MH⁺ 1201.6), DVQAWIRG*C (SEQ ID NO. 18) (MH⁺ 828.7),providing further evidence that disulfide bonds are broken duringpyrolysis. This valuable feature of pyrolysis-induced protein digestioneliminates the disulfide bond breaking step that usually involves andextra and time-consuming step necessary prior to enzymatic and chemicaldigestion methods¹⁶. In an enzymatic or chemical digestion procedure,cleavage of disulfide bonds commonly involves chemical digestionprocedure, cleavage of disulfide bonds commonly involves chemicalreduction of disulfide bonds (R—S—S—R¹) to thiol groups (R—SH) with theaddition of dithiothreitol, followed by alkylation of the reducedcysteines to prevent the residues from reforming their bonds throughoxidation¹⁶. The whole process needs 30 minutes to several hours,further adding to the total analysis time required for proteinidentification.

To test the ability of the pyrolysis-induced specific-cleavage atcysteine and to break disulfide bonds, more peptides and proteinscontaining cysteine residue(s) were examined.

Two more peptides containing cysteine residue(s) were tested:Antioxidant peptide A, PHCKRM (SEQ ID NO. 8) (FIGS. 18A and 18B), andSomatostatin 14, AGCKNFFWKTFTSC (SEQ ID NO. 9) (disulfide bond betweenthe two cysteines) (FIGS. 19A-C).

FIGS. 14 and 15 illustrate that pyrolysis indeed induces the two testedpeptides to cleave at the N-terminus of cysteine. For peptideAntioxidant A, the C-terminal cleave at the N-terminus of cysteine. Forpeptide Antioxidant A, the C-terminal side peptide fragment aftercleavage at cysteine is observed at m/z 505 and the MS/MS confirms itssequence. Furthermore, the mass calculation and MS/MS data show that theC-terminal side peptide fragment after C-cleavage has a modification of32 Da mass less than the original cysteine residue. Recall that in theprotein lysozyme, the N-terminal side peptide fragment after C-cleavagepossesses a modification of 1 Da mass less than the original, unmodifiedresidue

In the spectra of the pyrolyzed peptide Somatostatin 14, cleavages atboth of the two cysteines take place, both of the C, N terminal peptidefragments are present and both of the mass modifications (−32 and −1)are observed, further increasing the validity of the C-cleavage rule.Again, the sequences of the specific cleavage framents (m/z 1535.6 and1375.6) are confirmed by MS/MS. Moreover, the disulfide bond between thetwo cysteins proved to be broken by pyrolysis from the two fragments ofm/z 1535.6 and 1375.6.

Example 6 Pyrolytic Digestion of Proteins and their Identification

More proteins containing just cysteine (insulin bovine) and containingboth cysteine and aspartic acid α-lactalbumin (bovine milk), albumin(chick egg white), cytochrome C (horse heart)) were tested. Also, theirMS/MS date were used to identify the protein via database search—Mascotand Sequest V.3.3.1 (Thermo, San Jose, Calif.).

First, the date search was performed on the protein lysozyme. The MS/MSdata of ions at m/z 605, 828, 1201, 1327 and 1434 were manuallyextracted into a “*.dta” format file (format used by most databasesearches), which contains the precursor ion's mass and charge, and thefragmentation ions' mass and intensity. The searching parameters are asfollows and used for all the following searches. Database: MSDBTaxonomy: All entries; Enzyme: None; Allow up to 1 missed cleavages; Nomodifications; No quantitation; Peptide tol.: ±2 Da; MS/MS tol: ±1.5 Da;Peptide Charge: 1+, 2+, 3+; Monoisotopic; Data format: Sequest (.DTA);No precursor; Instrument: Default; No error tolerant; No Decoy; Reporttop Auto hits.

FIGS. 20A-C show the Mascot search results of lysozyme. Protein Lysozymeis successfully identified with a very high score.1. Recall that a scoreof 52 was obtained when ion m/z 1201 alone was used in the search. Inthis search five of the pyrolysis DC cleavage products are applied, thescore jumps to as high as 107, further increasing the identification'sspecificity. Moreover, the sequences of all of the fragment ions arecorrectly shown with the corresponding −1 Da modification (as the errorof the match or “Delta” value. This is the case because the web-basedfree access algorithm cannot take into account user-specified chemicalmodifications. A licensed version of this software is needed for thisability). The searching results of lysozyme are very encouraging,showing that a simple process as high temperatures heating or pyrolysishas the ability to digest proteins with site-specificity and thereforeidentity the protein via bottom-up proteomics. Furthermore, because thefree version of Mascot used cannot allow editing our own “enzyme”, the“no enzyme” option was selected. We believe a much better and morespecific search result could be obtained if an accurate “enzyme” (namelythe pyrolysis cleavage before C and after D with a all chemicalmodifications) were used.

Mass spectral data for lysozyme (containing full scans and MS/MS scans)was searched by another searching engine, Seaquest. Sequest is analgorithm that searches within a database containing peptidefragmentation spectra from known proteins for matches to spectraattained in the experiment^(17,18). In the Sequest search, an in silico“enzyme” was created within the BioworksBrowser™ to perform thepyrolysis digestion at D and C residues. However, in establishing thesearch parameters, “partially cleavage at either side” was selected,because of limiting flexibility in the “enzyme” editing. That is, thesoftware does not allow to generate an in silico enzyme which cleavagesat different sides of two residues, for example, to cleave at N-terminusor C and C-terminus of D. The other parameters used in the searchinclude up to 10 missed cleavage sites, group scan of 50, minimum groupcount of 1, minimum ion count of 12, the charge state option was set toauto, and the C-terminal peptide variable modification set as −1. Theabove parameters are used for all the following searching, unlessotherwise specified. The Sequest search of the entire databasesuccessfully identified the protein lysozyme with a probability score of6.3E6 and Xc score of 36 and both of the two scores are very good.However, only two (1328, 1435) of the 5 expected fragments aredetected/identified by Sequest, compared to 5 out of 5 found by theMascot database search.

The same Sequest search is conducted to lysozyme, but within thedatabase of chicken proteins. The probability score of 1.2E5 and abetter Xc score of 56 gained. Furthermore, 4 (828, 1201, 1328, 1435) ofthe 5 expected fragments are identified by Sequest, showing a bettersearch result if the protein source can be narrowed down.

FIGS. 21A-C show the ESI full scan mass spectrum of the protein insulinafter pyrolysis and the MS/MS mass spectra of m/z 566 and 778, thesodiated C-cleavage products confirm the sequences of both fragmentsanalyzed. The sequence coverage for pyrolysis DC cleavage of insulin is25.5%. Also, the first disulfide bonds connecting the two chains and theintra-chain A disulfide bond are broken. However the C-terminal sidepeptide.

The MS/MS data of ions at m/z 778 and 566 were manually extracted into a“*.dta” format file. For the sodiated ions, the value of 22 wassubtracted to the precursor ion's mass and each of the fragmentationions' to change them to protonated ions, but keep the intensityunchanged. FIG. 22 shows the Mascot search result of insulin using theMS/MS data of ions at m/z 778 and 566, showing the protein insulin issuccessfully identified, and both of the two fragment peptides aredetected.

Sequest searching was also performed on this protein; however it was notsuccessfully identified. Probable reason is that Sequest has problemdealing with small peptides, just as the ion m/z 605 is not identifiedto be part of lysozyme.

FIGS. 23A-F show the ESI full scan of protein α-lactalbumin (bovinemilk) after pyrolysis and the MS/MS spectra. The sequence coverage is upto 60.2%. MS/MS confirms their sequences.

The MS/MS data of prominent ions at m/z 1087, 599, 1493.7, 1403.6,1693.7, 617, 852, 936, 1511, 1712.6 were manually extracted into dtaformat files, which were used for the Mascot search. The Mascot searchresult successfully identified the protein α-lactalbumin with a veryhigh score of 130 (greater than 78 is significant), and 6 peptidefragments are detected. However, the very prominent peak at m/z 1087,corresponding to LC³²SEKLDQWL*C (SEQ ID NO. 19), is not detected by thesoftware, again due to limitations in setting up an in silico “enzyme”that accounts for the −32 modification of the N-terminal side peptidefragment in the free web-based version of Mascot.

Sequest result searched against the bovine database showed thesuccessful identification of the protein α-lactalbumin. Again, the veryprominent peak at m/z 1087, is not identified by the Sequest, due to thelack of proper accounting of chemical modifications by the data basesearch. However, the Sequest search against the whole database did notresult in the identification of the protein. Careful inspection showsthe peptides in the protein list are not generated according to the DCcleavage rule, which may be attributed to not being able to create thedesired in-silico pyrolyutic “enzyme” in Sequest (namely, cleavage atbefore C and after D at the same time). Hence, it is expected that abetter search outcome will result if using an appropriate in silicolpyrolytic enzyme.

FIG. 24 shows the ESI full scan mass spectrum of the protein albumin(chicken) after pyrolysis and the corresponding MS/MS mass spectra. Thepeaks at m/z 681 and 1821.7 correspond to the cleavage products at D.C.corresponding to the sodiated DHPFLF*C (SEQ ID NO. 20) and protonatedDAASVSEEFRADHPFLF*C (SEQ ID NO. 21), and their sequences are confirmedby MS/MS analysis. However, MS/MS data of the strongest peak at m/z1032.6 seems to indicate to the cleavage product of sequenceGSIGAASMEF*C (SEQ ID NO. 22), however, with a mass modification of +64.the MS/MS data show the b-ion series in the CID<but the y-ion series isnot. Further investigation is needed to illustrate its structure.

Mascot analysis of the MS-data was unable to identify this protein. Theunknown +64 modification of the peptide is partly suspected to causethis uncertainty. Subsequent Sequest search against the chicken databaseaccounting for the variable modification of −18 at the C-terminus ofpeptides shows albumin chicken is identified. However, Sequest searchagainst the entire database did not show the identification of albuminchicken.

The same experiment was conducted on the protein cytochrome C (horseheart). The only pyrolysis D, C cleavage product observed is an ion atm/z 1263, corresponding to the peptide of sequence DLIAYLKKATNE (SEQ IDNO. 23) (FIG. 25). the dta file of ion m/z 1263 is applied for Mascotsearch shown in FIG. 26. It shows the peptide fragment is a sequence tagand cytochrome C is identified. Also, Sequest search against the entiredatabase shows the identification of cytochrome C by identifying thesequence tag.

In summary, at this point, results presented demonstrated that pyrolysisfor 10 seconds and up to 220° C. induces peptides and proteins to cleavesite-specifically at the N-terminus of C and at the C-terminus of D.Moreover, sequence information of the original protein is retained afterthe pyrolysis process and the sequence tags can be detected by MS/MS. Inaddition, the MS/MS data can be used for successful database search(Mascot and Sequest) to identify the protein. However, limited successin protein identification has been achieved by this method since we areunable to design the appropriate pyrolytic in silico “enzyme” andaccounting for the proper chemical modifications. Finally, some of theexpected DC cleavage peptides are not observed in the ESI analysis.Considering that some of these peptides might be suppressed due to theionization suppression effects commonly present in ESI of complexmixtures, an HPLC separation is coupled online prior to the MS analysisto investigate this possibility.

Example 7 HPLC-Ms Analysis of Pyrolyzed Proteins and Database Search

To minimize the ion suppression effect in ESI, pyrolyzed proteins weresubjected to online HPLC separation prior to ESI-MS analysis. Thesoftware will automatically perform a full-scan mass spectrum followedby three data dependent product ion mass-spectral scans of the mostintense precursor ions (a.k.a., “big-three” scan).

FIG. 27 shows the chromatogram and the Sequest search result ofpyrolyzed lysozyme. The DC cleavage peptide fragments m/z 605, 828,1327.6, 1435.6 (also observed in direct injection) were detected at time15.6, 30.2, 35.7 and 39.4 min respectively (1201 not detected). TheSequest search against the chicken database shows lysozyme is identifiedand the identified peptides are produced following the DC cleavage rule.Still no more expected fragments are observed. Searching against theentire database did not yield the identity of lysozyme. Converting theRAW file into m/z data file, followed by a Mascot search also failed togive out the correct protein id. Because of the large quantity of theMS/MS data generated, it is not possible to manually extract them to adta file for Mascot search.

In the case of the protein albumin (chicken), it is identified bySequest search against the chicken database, but not against the entiredatabase.

The same HPLC-MS experiment was conduct to protein alactalbumin. FIG. 28illustrates its chromatograph and full MS scans at time 22,58 min and39.17 min showing the peptides eluting out of column. However, theSequest search did not yield its identity.

To sum up, HPLC separation prior to ESI analysis does not seem to helpidentify more expected peptide fragments as anticipated. It seems thatthe limiting factor in the analysis is the inability of the Sequestsearch engine, as mentioned before, to design the pyrolytic in silicoenzyme with the appropriate cleavage sites and chemical modifications.

Example 8 Application to Bacterial Identification

At this point, it is demonstrated that pyrolysis can used as a proteindigestion method to identify proteins by database search. In thissection, this methodology is extended to the identification of bacteriavia bottom-up proteomics using pyrolysis as the “enzyme”.

FIG. 29 shows the full ESI mass spectrum scan of pyrolyzed samples of E.coli and S. Aureus via direct infusion ESI-MS. FIG. 30 shows theon-probe DESI-MS (on-probe DESI is presented in detail in Chapter 3)full scan of pyrolyzed bacteria whole cells Salmonella typhimutium, andPseudomonas aureginosa. The full scans of these bacteria show there areno prominent peaks of peptides detected, majorly because of the ionsuppression effect and high complexity of the sample produced (afterwhole cells were pyrolyzed). To circumvent this limitation, HPLCseparation prior to ESI analysis was conducted on these samples.

The HPLC-MS experiment was conducted on the bacteria E. coli. FIG. 31illustrates its chromatogram and full scan mass spectra at retentiontime 25.34 min and 25.85 min as examples showing the peptides elutingout of column. Also good MS/MS data are obtained for each peptideeluting out of column at different times. The Sequest search in bacteriadatabase (FIG. 31) did give out some of its possible protein originsbeing from E. Coli, however not with high ranks (the highest is #57).Upon further inspection, the peptides detected for all the proteins inthe result list were not generated according to the DC cleavage rule(namely generated from cleavage before C and after D). Again, eventhough high S/N tandem mass spectra of peptides are obtained from thepyrolysis of intact bacteria, a capable search engine is still limitingfactor to perform a successful database search for bacteriaidentification.

The same experiment was done to bacteria S. aureus, and itschromatograph and full scan mass spectra at retention time 13.43 min and33.32 min (FIG. 32) showing the peptides eluting out of column. Justlike E. coli, the Sequest search in bacteria (FIG. 32) did give out oneof its possible protein origins being S. aureus; however, not with ahigh rank (#42). Again, the peptides detected for all the proteins inthe result list were not generated according to the DC cleavage rule(namely generated from cleavage before C and after D).

In summary, due to the complexity of the pyrolyzed bacteria, full ESI-MSanalysis does not show any peptide peaks and thus HPLC is required priorto entering the mass spectrometer. In HPLC-ESI-MS, peptide fragmentsfrom pyrolysis are eluted from HPLC column and are detected by MS. Theprecursor ions and their MS/MS data are used for Sequest databasesearching, which shows some of the proteins are actually from the testedbacteria. However, the identified peptides by Sequest are not producedfollowing the DC cleavage rule, because of the software's enzyme designlimitation. Thus, a capable search engine is needed to perform thedatabase search, to verify if pyrolysis can be applied for bacteriaidentification.

REFERENCES

-   (1) Baldwin, M. A. Mol. Cell. Proteomics FIELD Full Journal Title:    Molecular and Cellular Proteomics FIELD Full Journal 2004, 3, 1-9.-   (2) Kellner, R. L., F.; Meyer, H. E. Microcharacterization of    Protein, 2 ed.; Wiley-VCH: Weinheim, Germany, 1999.-   (3) Corey, E. J.; Haefele, L. F. Journal of he American Chemical    Society 1959, 81, 2225-2228.-   (4) Iwasaki, H.; Cohen, OL. A.; Witkop, B. J. Am. Chem. Soc. 1963,    85, 3701-3702.-   (5) Lewis, R. Elastomeric Proteins 2003, 136-151.-   (6) English, R. D.; Cotter, R. J. J. Mass Spectrom. 2003, 38,    296-304.-   (7) Yao, Z-P.; Afonso, C.; Fenselau, C. Rapid Commun. Mass Spectrom.    2002, 16, 1953-1956.-   (8) Anhalt, J. P.; Fenselau, C. Anal. Chem. 1975, 47, 219-225.-   (9) Meuzelaar, H. L. C.; Haverkamp, J.; Hileman, F. D. Techniques    and Instrumentation in Analytic Chemistry, Vol. 3: Pyrolysis Mass    Spectrometry of Recent and Fissil Biomaterials, 1982.-   (10) DeLuca, S.; Sarver, E. W.; Harrington, P.; Voorhees, K. J.    Anal. Chem. 1990, 62, 1465-1472.-   (11) Basile, F.; Voorhees, K. J.; Hadfield, T. L. Applied and    Environmental Microbiology 1995, 61, 1534-1539.-   (12) Hendricker A. D., V. K. J. Journal of Analytical and Applied    Pyrolysis 1996, 36, 51-70.-   (13) Meetani, M.; Basile, F.; Voorhees, K. J. Appl. Anal. Pyrolysis    2003, 68-69, 101-113.-   (14) Thomya, P.; Wesdemiotis, C.; Erdodi, G.; Kennedy, J. P.,    Seattle, Wash., May 29-Jun. 1, 2006 2006; American Society for Mass    Spectrometry.-   (15) Sato, M.; Hida, M.; Nagase, H. Journal of analytical toxicology    2001, 25, 304-309.-   (16) Hauser, N. Ph.D. dissertation, University of Wyoming 2008,-   (17) Yates, J. R., III; McCormack, A. L.; Eng, J. Analytical    Chemistry 1996, 68, 534A-540A.-   (18) MacCoss, M. J.; Wu, C. C.; Yates, J. R., III Analytical    Chemistry 2002, 74, 5593-5599.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art that have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

We claim:
 1. A method of analyzing a peptide sample containing asparticacid and cysteine, comprising the steps of: (a) heating the peptidesample to between about 180° C. and about 250° C., in a period ofbetween about 5 seconds and about 30 seconds to cleave the peptide atboth the aspartic acid and cysteine site-specific locations; (b)electrospraying the digested sample with a solvent to produce desorbedions of components of the digested sample; and (c) detecting thedesorbed ions.
 2. A method as described in claim 1, wherein the step ofdetecting the desorbed ions is by mass spectrometry.
 3. A method asdescribed in claim 1, wherein the solvent is free of cationizing agents.4. A method as described claim 1, wherein the method is performed atatmospheric pressure.
 5. Apparatus for analyzing a peptide samplecontaining aspartic acid and cysteine, comprising: (a) a heating elementhaving a surface on which the sample is deposited and which heats thepeptide sample to between about 180° C. and about 250° C., in a periodof between about 5 seconds and about 30 seconds to cleave the peptide atboth the aspartic acid and cysteine site-specific locations; (b) anelectrospray device that subjects the digested sample to a solvent sprayto produce desorbed ions of components of the digested sample; and (c) adetector for detecting the desorbed ions.